7.1 INTRODUCTION:
T he candidate gene approach has been successfully used in the identification o f many disease genes in general, and m ost o f the retinal disease genes (e.g. rhodopsin and peripherin) in particular. In contrast to positional cloning this approach offers a short cut to the process o f disease gene identification. Screening o f a suitable candidate gene can lead to the identification o f th e disease causing m utation or exclusion o f such a gene from disease causation. In addtion to being sim ply in the right place or genetic interval (positional candidate), candidate genes for retinal diseases can be genes which are involved in the visual transduction pathw ay o r structural proteins involved in the form ation or function o f the retina. A lternatively they could be m em bers o f a gene fam ily another m em ber o f which is known to cause a related phenotype. O ther candidates could be genes which when mutated cause sim ilar phenotype in animal m odels o f hum an disease (eg. peripherin/R D S and PDEB), or genes which are know n to cause phenotypes th at could be allelic to the disease under study. T he term "candidate" is also used to describe candidate loci identified by linkage analysis, and w here genes are not yet identified. C andidate loci can be excluded by linkage analysis in test fam ilies o f m arkers which o riginally showed linkage to related disease phenotypes. T he last concept o f candidate loci does not apply to the subject o f this chapter.
7.2 A CANDIDATE GENE FOR R P ll: PRKCG
T he R P l 1 locus has been mapped to chrom osom e 1 9 q l3 .4 (chapter 5). T he disease interval has been estim ated as 5cM between D19S180 and AFM cOOlybl (fig 6.2 ). T his is a relatively larg e genetic interval, for which physical mapping could be a lengthy process. T he candidate gene approach is preferable in such cases if a suitable candidate can be identified. C hrom osom e 19 is particularly gene rich (Craig and B ick m o r^ . Searching the genom e data base for genes in that region identified a num ber o f genes most o f which code for proteins w hose functions w ould appear to be unrelated to any mechanism that could result in visual im pairm ent o r RP.
However, the gene encoding the isoenzyme pro^in kinase C gamma (PRKCG) has been
previously mapped to chromosome 19ql3.2-ql3.4 (Coussens û/., 1986, Johnson era/,, 1988)
and could be considered as a candidate gene (see below).
7.3 W hat is PRKCG:
Protein kinase C (PKC) is a multifunctional family of closely related serine/threonine protein kinases that are activated by diacylglycerol (DAG) (Hug and Sarre, 1993; Nishizuka, 1992). PKCs function in a wide variety of cellular processes, such as membrane receptor signal transduction, control of gene expression and control of cell division and differentiation (Hug and Sarre, 1993; Nishizuka, 1988). PKC isoenzymes are ubiquitously distributed, but they are
expressed in a tissue-specific manner (Ohno et al., 1987; Brandt et al., 1987; Westel et al.,
1992)
Initially three related members of this gene family, termed a, jS, and 7, were cloned from
bovine brain cDNA libraries and homologoes were obtained by screening a human cDNA
library with the unique portions of the bovine clones (Coussens et al., 1986). The three human
genes were assigned to different autosomes on the basis of segregation in human-rodent hybrid
panels and by in situ hybridization. The ot gene was assigned to chromosome 17, the jS gene to
chromosome 16, and the 7 gene to chromosome 19 (region 19ql3.2-ql3.4). These members of
the PKC gene family were found to be closely related at both the amino acid and the nucleotide levels. Later cDNAs were cloned from many other PKC isoforms, mostly from brain cDNA libraries (reviewed by Hug and Sarre, 1993). Southern hybridization analysis suggested that an even larger number of PKC genes may exist, and the diversity within this family of proteins may be further increased by alternative splicing in at least some of these genes (for example /3I and jSII isoenzymes; Hug and Sarre, 1993).
PKCs were divided into two main groups: the Ca^^-dependent or conventional PKCs (cPKCs) and the Ca^^-independent or novel PKCs (nPKCs) (Hug and Sarre, 1993). The earlier
isolated PKC isoforms ot, /SI, /SII and 7 belong to the Ca^^-dependent group, and the newly
identified isoforms b, e, f, 1/, and 6, to the Ca^^-independent group. The primary amino acid
structure of PKCs deduced from the cDNA sequences, can be divided into conserved (presumably functional) domains termed C1-C4, which are separated by variable regions termed
V1-V5, the function of which is not yet known (Coussens et al., 1986; fig 7.1). All PKC
A candidate gene fo r R P ll: PRKCG V I H i n g e r e g i o n C l V 2 C 2 V 3 C 3 V 4
. i i
C y s t e i n e - r i c h r e p e a t i i A T P - b i n d i n g f i t eA
A
A
A
A
R e g u l a t o r y d o m a i n C a t a l y t i c d o m a i n Figure 7.1:(Figure and legend are from Hug and Sarre. 19931.
Domain structure o f PKC isoenzymes. All PKC isoenzymes consists o f constant (C) and variable (V) regions. The cys-rich repeats in the C l region and the ATP binding site in the C3 region are indicated by arrow heads. The arrow points to the hinge region in the V3 dom ain which separates the regulatory from the catalytic domain.
regions C3-V5 have been defined in all PKC isoenzymes as the catalytic domain, which is separated by the V3 region from the N-terminal regulatory domain. In addition to Ca^^ dependence, the two groups of PKC vary in their regulatory domain (fig 7.1). The nPKCs lack the C2 domain. This domain was thought to contain the Ca^^ binding site of the cPKCs, but no sequence motif that represents a known Ca^^-binding site, such as the classical E-F hand binding motif, were identified (Hug and Sarre, 1993).
7.4 WHY PRKCG IS A GOOD CANDIDATE GENE FOR R P ll:
The following three sections outline the reasons for considering PRKCG as a candidate for R P ll.
7.4.1 It maps in the right genetic interval:
The gene for PKCy isoenzyme was mapped to 19ql3.4 region (Coussens et al., 1986;
Johnson et al., 1988; this study). Haplotype analysis of the Mspl polymorphism detected by this
gene showed that PRKCG is localized between D19S572 and AFMcOOlybl (see section 5.2.6). This is the interval which was found to segregate with the R P ll phenotype in ADRP5. This colocalization of the two loci (RPll and PRKCG) suggests that PRKCG could be a candidate gene for R P ll.
7.4.2 PKC phosphorylates rhodopsin in the ROS:
The function of PKCy is not yet clearly defined. However PKCs, in general, have been shown to be expressed widely in the retina and particularly in the outer segments of photoreceptors (Newton and Williams, 1993). Phosphorylation by protein kinase C results in the desensitization of a large number of receptors, suggesting an important function of this group of isoenzymes in the adaptation of cells to extracellullar information (Newton and Williams, 1993). Examples of membrane receptors whose function is modulated by PKC include EGF (epidermal growth factor) and G-protein linked receptors such as the ^-adrenergic receptor
(Kelleher et al., 1984; Fearn and King, 1985). The observation by Newton and Williams (1991,
1993) that protein kinase C phosphorylates rhodopsin in a light-dependent manner suggests the involvement of PKC in desensitizing rhodopsin. While RK (rhodopsin kinase) activity (see section 1.4.6) predominates at higher light levels, PKC activity was suggested to predominate at low levels. PKC catalyzes the phosphorylation of rhodopsin on a carboxyl terminal domain
A candidate gene f o r R P ll: PRKCG
phosphorylation is independent of the activation state of rhodopsin (Newton et al., 1994). This
PKC-catalyzed phosphorylation of rhodopsin has been shown to uncouple the receptor from
transducin in vitro (Kelleher and Johnson, 1986), suggesting that the phosphorylation deactivates
the rhodopsin molecule. In another study, PKC immunoreactivities were detected in cone photoreceptors of the rat retina, where PKC was suggested to phosphorylate cone visual
pigments (Ohki et al., 1994).
7.4.3 PKC mediates a retinal degeneration in Drosophila:
7.4.3.1 Visual transduction in drosophila:
The invertebrate visual cascade, like that in vertebrates, begins with the light activation of
rhodopsin molecules and subsequent activation of a G-protein (Smith et al., 1991a). The effector
molecule that mediates the vertebrate visual cascade is a G protein-activated cGMP-PDE (see section 1.5), which lowers cGMP concentration in response to light. This leads to the closure of channels and a drop in calcium concentration. In drosophila, a G protein-coupled phospholipase C (PLC) functions as an effector. PLC catalyzes hydrolysis of
phosphatidylinositol bisphosphate (PlPg) into the second messengers inositol trisphosphate (IP3)
and diacylglycerol (DAG). IP3 appears to be a mediator of photoreceptor cell activation (Fein, 1986). This activation leads to the opening of channels and an increase in the cytosolic calcium concentration. In contrast to vertebrates the end result of this activation is the depolarization rather than the hyperpolarization of the photoreceptor cell (fig 7.2). DAG was suggested to function in the feedback regulation through the activation of eye-PKC of drosophila, see below (Nishizuka, 1992).
7.4.3.2 Drosophila retinal degeneration mediated by eye-PKC:
A Drosophila melanogaster PKC gene known as eye-PKC was isolated and characterized
(Schaeffer et al., 1989). This gene was found to map to position 53E on the second
chromosome, within 50kb of another previously reported Drosophila PKC gene (Rosenthal et
al., 1987). It was found to be exclusively expressed in photoreceptor cells and was related to
the cPKC genes (Schaeffer et al., 1989).
The visual systems of both vertebrates and invertebrates are able to modulate their sensitivity to light in a process known as light adaptation (see section 1.4.1). This adaptation appears to be mediated through the changes in the calcium concentration within the
Figure 7.2:
(Figure and legend are from Smith et al.. 1991hl
h v
\
R - M - P O A r r e s l i n ^ ^ G c 3 y - G D P cl T / ^ n o r c A , G T P G D P K + A T P ( - ) P3 4- D i a c y l g l y c e r o lI
\
(çZD
( c y c l i c G M P ) ? e y e - P K C Ca+ 2 In'.racellular Extracellular Na+ C a+ 2 M o d e l o f e y e - P K C f u n c t i o n . A b s o r p t i o n o f a p h o t o n o f lig h t c au ses a c o n f o r m a t i o n a l c h a n g e in t h e r h o d o p s i n m o l e c u l e ( R ) aj'.d activates its c a t a l ) t i c p r o p e r t ie s . A c t i v e m e t a r h o d o p s i n ( M * ) catal)ctes G p r o t e in a c tiv a t i o n . T h e G p r o t e i n e x c h a n g e s G D P f o r G T P a n d releases t h e in h ib it o r y P7 s u b u n i t s . A c t i v e G p r o t e in cat;d )'zcs t h e a c t i v a t i o n o f th e nerpAL-cncoded P L C , a n d P L C hydrol)"zes P iP ^ i n t o I P3 a n d D A G . T h e I P3 is released fr o m t h e m e m b r a n e a n d d if f u s e s t o r e c e p t o r s l o c a t e d o n t h e s u b r h a b d o m e r i c c is t e r n a c ( S R C ) . T h i s b i n d i n g is t h o u g h t t o r eleas e in tr acell u la r c a lc iu m fr om S R C , w h i c h a p p e a r s to b e i n v o l v e d in e x c i t a t i o n . C y c lic G M P has also b e e n i m p l i c a t e d as a p o s s i b l e i n t r a c e llu la r m e s s e n g e r m e d i a t i n g e x c i t a t i o n {5S, 5 9 ) .E x tr a c e llu la r s o d i u m a n d c a l c i u m t h e n e n t e r t h e cell t l i r o u g h th e lig h t-